Project Summary/Abstract Proteome homeostasis (proteostasis) is essential for cellular life and is maintained by an array of cellular components that regulate the synthesis, folding, solubility and degradation of proteins throughout the proteome. Optimal cellular function is achieved when these proteostasis network (PN) components work collectively to calibrate the levels of correctly folded, and thus functional proteins, to meet cellular demands and prevent the formation of toxic protein aggregates. Many inherited forms of conformational diseases (CD), including many neurodegenerative diseases, are caused by gene mutations that result in non-native conformations (folding) of a single underlying protein, which increases the propensity of these proteins to aggregate. Cellular dysfunction in the context of the overexpression of these conformationally compromised aggregation-prone proteins has been linked to proteostasis impairment (PI). However, the timing and nature of the cellular events that lead to PI in CD remain elusive. A growing, but limited body of evidence suggests that a phenomenon termed chaperone titration (CT) is mechanistically linked to and may initiate PI in CD. Chaperones are essential PN components as they regulate the de novo folding or refolding of denatured client proteins, prevent, resolve or remodel client aggregates and target terminally misfolded or aggregated clients for degradation. The CT model posits that increasing preoccupation of chaperones with one ?compromised? client results in the titration of these chaperones away from their other clients leading to impaired folding and/or degradation of these clients. The goal of this proposal is to determine the extent and timing of CT in response to the accumulation of a conformationally compromised aggregation-prone protein. This goal was unattainable until now due to a previous lack of biochemical tools to globally assess multiple metrics of impaired chaperone maintenance and a lack of models in which the unfolding of a single protein could be induced with the acuteness and synchrony required to accurately test this model. To do this, the proposed studies will utilize a proteomics toolkit designed to detect changes in multiple protein metrics consistent with impaired chaperone maintenance and a novel cell model in which the unfolding and aggregation of a single protein (DD) can be induced with unprecedented acuteness and synchrony (within sec to min) in a cell population upon removal of a stabilizing ligand. In Aim 1, the proteome will be screened to identify the putative ?normal? clients of the chaperones that are titrated away by DD using this toolkit which measures alterations in protein ubiquitination, total levels, degradation rates and recruitment to DD aggregates. In Aim 2, these alterations will be validated using orthogonal biochemical analyses. In Aim 3, the extent of CT will be elucidated through experiments designed to definitively identify chaperones that are titrated away by DD and the ?normal? clients of these chaperones (putatively identified in Aims 1 and 2) affected by their titration. This research will advance our understanding of the molecular pathogenesis of CD and the interconnectedness of chaperone-client networks.